Cyclic adenosine monophosphate (cAMP, also known as cyclic AMP or 3′-5′cyclic adenosine monophosphate) is a second messenger involved in many biological processes in many organisms. For example, cAMP plays important roles in intracellular signal transduction in many organisms. The cAMP-dependent signal transduction pathway is a G protein-coupled receptor (GPGR)-triggered signaling cascade, which mediates various biological processes, e.g., glycogen, sugar, and lipid metabolism.
The bioluminescence resonance energy transfer (BRET) methods are based on resonance energy transfer between a light-emitting enzyme and a fluorescent acceptor. Bacart et al., Biotechnol. J. 3:311-324 (2008); Barak et al., Mol. Pharma. 74:585-594 (2008). Because the BRET technology is cell-based and non-destructive, it is well suited for proteomics applications, including studies on protein-protein interactions. However, to reduce cAMP detection background and obtain better separation of the donor and acceptor energy emission peaks, improvements in BRET assays are needed.
Glucagon-like peptide-1 receptor (GLP-1R) signaling is an established therapeutic target for type 2 diabetes. In addition to human pancreatic islet β cells, GLP-1R is expressed in a wide array of tissues, including lung, heart, kidney, blood vessels, neurons, and lymphocytes (1-4). Mice deficient in GLP-1R expression or with blunted GLP-1R function show impairment of physiologic features not limited to glucose homeostasis but also include learning and memory (4). Clinical trials targeting GLP-1 signaling to treat non-metabolic diseases include those for psoriasis, heart disease, and neurodegenerative diseases (5-7). Despite encouraging outcomes with GLP-1 analogs in reducing myocardial infarct size in acute coronary occlusion (7) and improving clinical symptoms in patients with Parkinson's disease (5), the mechanisms of physiological regulation of GLP-1R signaling beyond energy homeostasis remain largely unknown.
GLP-1 is an incretin peptide hormone derived from post-translational processing of the precursor proglucagon in intestinal L cells (8). On food intake, the biologically active forms of GLP-1 (7-36) amide and GLP-1 (7-37) are secreted, thus increasing the basal plasma level by 3- to 4-fold, to maintain normoglycemia by enhancing glucose-dependent insulin secretion and suppressing glucagon function (8,9). Circulating GLP-1 has a short plasma half-life of only a few minutes due to renal clearance after rapid enzymatic inactivation by a plasma enzyme, dipeptidyl peptidase 4 (DPP 4) (10). Other cells outside of the gut shown to produce GLP-1 include pancreatic α cells and neurons in the localized area of the brain stem (4,11-14), but our knowledge of the physiological regulation of GLP-1 secretion by these cells is limited.
In the brain, GLP-1 is synthesized primarily by a discrete group of neurons located in the nucleus of the solitary tract (12). These neurons send abundant projections to other regions of the brain, including forebrain, hypothalamus, amygdala, stria terminalis, and thalamus, where GLP-1Rs are expressed; this neuronal circuit of GLP-1 signaling is considered relevant to satiety and energy homeostasis (2,11). GLP-1R is also expressed in neurons in the hippocampus (1) and dopaminergic neurons in substantia nigra (3)—where no known GLP-1-secreting neuron innervation is found (3,14). It has been suggested that the basal circulating GLP-1 level is the primary source of ligands accessible to GLP-1Rs in these brain regions and probably in the heart as well. Therefore, determining the mechanism by which basal level of GLP-1 can activate receptors in these brain regions is germane.
The well-delineated functions of GLP-1 are mainly mediated by activation of GLP-1R (4). GLP-1R, as a member of the class B G protein-coupled receptor (GPCR) family, is the only known receptor with high specific affinity for GLP-1. GLP-1R activation leads to two major signaling pathways, namely Gαs coupling and recruitment of β-arrestin to the agonist-occupied receptor; the former mainly leads to activation of adenylyl cyclase, with subsequent generation of cAMP (15), and the latter leads to receptor endocytosis and activation of extracellular signal regulated kinase (ERK) 1/2 signaling (4). In pancreatic β cells, the increased cAMP level is responsible for glucose-dependent insulin release (16) and contributes to maintaining glucose homeostasis. Thus, cAMP production is measured and used as GLP-1R-mediated functional response in properly designed assays.
GLP-1 receptor (GLP-1R) is expressed in many peripheral and neuronal tissues, and is activated by circulating GLP-1. Other than food intake, little is known about factors regulating GLP-1 secretion. Analysis of food intake-induced increase in GLP-1 level and subsequent activation of GLP-1R have provided some insights into the role of GLP-1R signaling in energy homeostasis. However, the short half-life and low basal level of circulating GLP-1 (7-36) amide do not permit assessment of the physiological relevance of GLP-1R signaling other than energy homeostasis.
Current GLP-1 analogue therapeutics requires frequent subcutaneous administrations, and leads to reduced compliance and high prices in developing area. Typically, the plasma level of active GLP-1 is around 5 to 10 μM in the basal state, quickly rises to 20 to 50 μM after oral glucose or meal and will slowly declines to basal level over 2 hours. However, GLP-1 analogue therapeutics usually require to maintain constantly a supra-physiological level of GLP-1 analogues, thus lead to activating GLP-1 receptors constitutively and may cause severe complications upon chronic treatment. Identification of novel compounds that modulate the endogenous GLP-1 receptor signaling pathways can lead to the development of new therapeutics useful in regulating blood glucose levels, thereby treating diabetes or disorders associated with the GLP-1 receptor.